Multidrug resistance (MDR), the principle mechanism by which pathogens manifest resistance to biologically active agents, is a major public health challenge. The multidrug resistance phenotype is dependent upon the expression of enzymes that degrade specific classes of cytotoxic agents, and/or molecular pumps that are capable of expelling exogenous agents from the cytoplasm of bacterial, fungal and mammalian cells. Moreover, the expression of MDR in bacterial, fungal and malignant cells poses obstacles for the process of drug discovery. Assays designed to screen compounds, or libraries thereof, may fail to reveal the presence of active constituents due to the presence of MDR in the cell lines of the assays. This phenomenon is certain to leave undiscovered new, and potentially highly beneficial, drug candidates. At the very least, the various mechanisms of MDR undermine the efficacy of known cytostatic and/or cytotoxic compounds.
Pathogenic bacteria continue to be a major source of disease in humans and animals. Improvements in minimizing bacterial contamination have made substantial inroads in eradicating or containing the spread of bacterial disease. Many bacterial infections can be effectively treated in the acute phase of infection with antibiotics. There are, however, a large number of diseases that escape early detection and don't respond to antibiotic treatment. Added to this is the possibility of misdiagnosis, inavailability of medical care, development of antibiotic resistant organisms or asymptomatic primary infection. Many bacterial and parasitic diseases are endemic to certain regions of the world; in particular, the tropics and sub-tropics. The problem is exacerbated in those areas suffering from inadequate water and sewage treatment. The need for new antibacterials is due predominantly to two factors: the spread of antibiotic resistance genes; and the posture adopted by major pharmaceutical firms during the 1970s and 1980s that the market for new antibiotics had permanently diminished.
Antimicrobial resistance continues to spread in nosocomial pathogens in acute care hospitals and other key settings of managed health care systems. Appropriate control measures for such resistant organisms depend, in part, on the pathways by which resistance has arisen. Unfortunately, these pathways differ greatly from organism to organism and setting to setting. Although the epidemiology of resistant organisms sometimes is similar to that of susceptible organisms of the same kind, in some situations it may be quite different. Bacterial MDR pumps that expel known disinfectants have made it more difficult to sterilize hospital settings. Likewise, bacterial MDR pumps have undermined the effectiveness of antiseptics and antibiotics.
Acquired resistance to chemotherapy is a major problem in treatment of cancer by conventional cytotoxic drugs. Tumors may initially respond well to chemotherapy but later become resistant to a variety of unrelated drugs, leading to relapse. MDR in malignant cells is largely dependent on the expression of one or the other or both of two genes: mdr1 and mrp. These genes encode transmembrane energy-dependent molecular “pumps” that expel a wide variety of anticancer agents from malignant cells (Grant et al., Cancer Res. 54: 357-361, 1994). The normal functions of these molecular pumps are not well defined, but both are expressed by hematopoietic cells; P-gp, additionally, has been shown to have a causal role in immunofunction (Gupta et al., J. Clin. Immunol. 12: 451-458, 1992). The MRP is a molecular pump initially found to be involved in MDR in lung cancer, and then later found to be expressed in other cancer types. This protein is overexpressed in certain tumor cell lines which are multidrug resistant but do not overexpress P-glycoprotein (Cole et al. (1992) Science 258:1650-1654; Slovak et al., (1993) Cancer Res. 53:3221-3225). P-gp is a molecular pump long known to be involved in producing multidrug resistance in many tumor types (Chin et al., Adv. Cancer Res. 60: 157-180, 1993; Cole et al., Science 258: 1650-1654, 1992; Grant et al., Cancer Res. 54: 357-361, 1994; Krishnamachary and Center, Cancer Res. 53: 3658-3661, 1993; Zaman et al., Cancer Res. 53: 1747-1750, 1993).
Studies in which inhibitors of bacterial MDR pumps or P-gp inhibitors were administered to resistant bacterial or malignant cells, respectively, have shown that such competitive inhibitors can increase the response of the target cells to cytotoxic pharmaceutical agents. For example, MDR malignant cells are rendered more sensitive, under the aforementioned conditions, to the cytotoxic agents without an equivalent increase in the sensitivity of surrounding normal tissues.
In a second approach to attenuating the multidrug pumps, either bacterial or mammalian, oligonucleotides, or analogues thereof, are administered that are designed to block at the level of translation the expression of the genes that code for the pumps. These antisense oligonucleotides have several features which make them clinically attractive. Success in the area of chemotherapy illustrates this point: There are reports in the literature of reduced drug resistance in cultured cell lines following treatment with oligonucleotides targeting MDR-1 mRNA. Thierry et al. (Biochem. Biophys. Res. Comm. 190: 952-960, 1993) report a oligonucleotide 15-mer that gave 95% inhibition of MDR-1 expression when encapsulated in liposomes; this effect was associated with a 4-fold increase in sensitivity of the tumor cells to doxorubicin. When this 15-mer was administered without liposomes, inhibition of MDR1 expression was only 40% of control values. Jaroszewski et al. (Cancer Comm. 2: 287-294, 1990) and Corrias and Tonini (Anticancer Res. 12: 1431-1438, 1992) both found inhibition with only one out of five candidate oligonucleotides. Jaroszewski et al. describe a phosphorothioate that gave 25% reduction in P-gp expression at 15 μM and 33% reduction at 30 1M when incubated with MCF-7/ADR breast cancer cells for 5 days. This reduction in P-gp expression was associated with a small increase in the doxorubicin sensitivity of the cells (20% increase in cell death when 10 μM of the oligonucleotide was used). Corrias & Tonini report a phosphodiester oligonucleotide that gave only a slight reduction in P-gp at 30 μM when incubated for 36 h with doxorubicin-resistant colon adenocarcinoma cells. The reduction in P-gp expression was associated with a significant increase in the in vitro sensitivity of the cells to the cytotoxic effects of doxorubicin (80% and 53% dose reductions in IC50, respectively).
The process of drug discovery can be hampered by the presence of MDR phenotypes in bacteria, fungi, and malignancies. These phenotypes may be expressed in the organisms and cell lines exploited for in vitro screening of new drug candidates; more importantly, over time the MDR phenotype can evolve “unnoticed” in a cell line used for in vitro assays. This situation would lead to false negatives, and thereby leave undiscovered potentially rewarding new leads. This situation impacts the discovery of new lead compounds; compounds that would otherwise be cytotoxic are made to appear inactive in assays by the underlying mechanisms of MDR. A means of reliably separating these variables promises to provide new lead compounds active against bacterial infections, fungal infections, and cancer. Additionally, similar logic may be applied to the discovery of so-called chemosensitizers, compounds that disrupt the MDR phenotype but are not themselves cytotoxic.